Amino acid changes in conserved regions of the beta-subunit of Escherichia coli RNA polymerase alter transcription pausing and termination.

Abstract

Control of transcription at pause and termination sites is common in bacteria. Many transcriptional pause and termination events are thought to occur in response to formation of an RNA hairpin in the nascent transcript. Some mutations in the 13-subunit of Escherichia coli RNA polymerase that confer resistance to the transcription inhibitor rifampicin also alter the response to transcriptional pause and termination signals. Here, we report isolation of termination-altering mutations that do not confer rifampicin resistance and show that such mutations occur predominantly in limited regions of the 13-subunit polypeptide. One region is between amino acid residues 500 and 575, which encompasses the locations of almost all known rifampicin-resistance mutations. Many termination-altering mutations also occur in two other regions: between amino acid residues 740 and 840 and near the carboxyl terminus of the I~-subunit (amino acid residues 1225-1342). Amino acid sequences in these three regions of the 13-subunit are conserved between prokaryotic and eukaryotic 13-subunit homologs. Several mutations that alter transcription termination in vitro affect amino acid residues that are identical in prokaryotic and eukaryotic RNA polymerase 13-subunit homologs, suggesting that they alter an important function common to multisubunit RNA polymerases. We propose that these three regions of the 13-subunit may contact the nascent RNA transcript, the RNA-DNA heteroduplex, or the DNA template in the transcription complex and that mutations in these regions alter transcription pausing and termination by affecting these contacts.

Folding of noncoding RNAs during transcription facilitated by pausing-induced nonnative structures

“…The wild-type E. coli RNA polymerase was purchased from USB (Cleveland, OH). The mutant E. coli RNA polymerase (␤: P560S, T563I) was a generous gift from Robert Landick (University of Wisconsin, Madison, WI) (21). Polyhistagged B. subtilis RNA polymerase was purified in a slightly modified protocol, as described (32).…”

Differences in community composition of bacteria in four glaciers in western China

“…More data on the meteorologic, physico-chemical, and biological characteristics of the surface snow and consistent ice core data of microbial analysis will be crucial for truly understanding the community dynamics of microorganisms in glacier ice. More sequence data based on multiple-loci genes such as recA (encoding the multi-functional DNA-binding protein involved in homologous recombination; Story et al, 1992;Kumar et al, 1993), gyrB (encoding the DNA gyrase B protein; Hallett et al, 1990), wzm (encoding the inner membrane protein; Reeves et al, 1996), and rpoB (encoding the β-subunit of RNA polymerase B; Landick et al, 1990) of the chromosome of microorganisms in glacier ice may provide the necessary detailed information on microbial biogeography, since the sequence data in this study was based on only a single 16S rRNA gene analysis.…”

Three Amino Acids in Escherichia coli CspE Surface-exposed Aromatic Patch Are Critical for Nucleic Acid Melting Activity Leading to Transcription Antitermination and Cold Acclimation of Cells

“…We therefore determined the effects of substitutions in CspE surface-exposed aromatic patch on transcription antitermination in vivo. The E. coli strain RL211 designed by Landick et al (21) contains the cat gene preceded by a strong -independent trpL terminator and is therefore sensitive to chloramphenicol. When transcription termination at trpL is reduced, the cat gene is expressed, and the cells become resistant to chloramphenicol.…”